وأيد هذا العمل من خلال منحة من المعهد الوطني لالتهاب المفاصل والأمراض الجلدية العظام والعضلات ، NIH RO1AR054458.

1. وسم hMSCs مع Endorem <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تزرع الخلايا لالتقاء 80 ٪ على الأقل 18 ساعة قبل وضع العلامات. </li><li style=";text-align:right;direction:rtl"> خلال هذا الوقت ، على استعداد وسائل الاعلام الوسم بإضافة Endorem إلى نموذج بجرعة 100 ميكروغرام الحديد / مل مصل خالية من وسائل الإعلام. </li><li style=";text-align:right;direction:rtl"> بعد تزرع الخلايا لالتقاء ، ويستنشق وسائل الإعلام والثقافة ، ويتم غسل الخلايا 1X مع برنامج تلفزيوني أو وسائل الإعلام المصل الحرة. </li><li style=";text-align:right;direction:rtl"> المقبل ، ويستنشق الحل شطف وإضافة وسائل الاعلام الوسم أعدت مسبقا. ثم يتم تحضين الخلايا عند 37 درجة مئوية ، 5 ٪ CO 2 لمدة 4 ساعات. </li><li style=";text-align:right;direction:rtl"> بعد 4 ساعات الحضانة ، ووضع العلامات وسائل الاعلام هو يستنشق وشطف الخلايا مع برنامج تلفزيوني وtrypsinized كالمعتاد. </li><li style=";text-align:right;direction:rtl"> trypsinized مرة واحدة ، يتم غسل الخلايا 3X مع برنامج تلفزيوني وطرد في 400rcf لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> بعد هذه الخطوة الغسيل ، ومعلق الخلايا ، وعدها ، المقررة للبقاء ، وتستخدم حسب الحاجة من أجل التجريب. </li></ol> 2. إعداد Agarose <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد أن تم تسمية الخلايا أن الوقت قد حان لإعداد حل agarose. </li><li style=";text-align:right;direction:rtl"> على نطاق وغرامة ، ويتم وزن 80mg من مسحوق agarose وإضافتها إلى 2ml من برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> ثم يتم هذا الحل اهتزت بلطف وتعقيمها. </li><li style=";text-align:right;direction:rtl"> بعد حوالي 40 دقيقة ، ويمكن أن تؤخذ في حل agarose السوائل من الأوتوكلاف. وينبغي قياس حجم وتعديلها وفقا لبرنامج تلفزيوني مع مسخن. </li><li style=";text-align:right;direction:rtl"> ثم يتم تبريد المحلول وصولا الى 42 درجة مئوية </li></ol> 3. خلق مازي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عندما حل agarose قد وصل إلى درجة الحرارة المطلوبة ، يتم الاعتماد على الخلايا وطرد للمرة الأخيرة في أنبوب إيبندورف 1.5ml. </li><li style=";text-align:right;direction:rtl"> بعد الطرد المركزي ، ويتم تجاهل طاف وتختلط مع خلايا DMEM من أجل التوصل إلى تركيز 30 × 10 6 خلية / مل. </li><li style=";text-align:right;direction:rtl"> الآن تختلط الخلايا التي تحتوي على 42 درجة مئوية agarose الدافئة. </li><li style=";text-align:right;direction:rtl"> وهناك معلومات ماصة الباردة قد يؤدي إلى ترسيخ agarose داخل الحافة ، ولذلك ينصح بأن يزج تسخين بواسطة pipetting الساخنة العقيمة PBS صعودا وهبوطا </li><li style=";text-align:right;direction:rtl"> الآن ، نفس الحجم من agarose كما اتخذت وسائل الإعلام حتى وخلطها بشكل تام مع الخلية وسائل الإعلام ، بحيث يتم تعليق إنشاء تعليق متجانسة. </li><li style=";text-align:right;direction:rtl"> الحفاظ على أنبوب إيبندورف داخل حمام ماء مسخن حين خلط بحيث agarose لا يبرد بما يكفي لترسيخ. </li><li style=";text-align:right;direction:rtl"> عندما وقف ومتجانسة ، وزرع الخلايا في العيب. </li></ol><img src="/files/ftp_upload/1793/1793fig1.jpg" alt="الشكل 1" /> الشكل 1. T2 المرجحة صور الاكليل SE (TR 4000 مللي / TE 18.27 مللي) من عينة الرضفة مع hMSCs مزروع في عيوب الغضروف.

<ol>
	<li>Mauck, R. L., Yuan, X., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+differentiation+and+functional+maturation+of+bovine+mesenchymal+stem+cells+in+long-term+agarose+culture.">Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture.</a> Osteoarthritis Cartilage. 14, 179-189 (2006).</li><li>Pittenger, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+potential+of+adult+human+mesenchymal+stem+cells.">Multilineage potential of adult human mesenchymal stem cells.</a> Science. 284, 143-147 (1999).</li><li>Rahfoth, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+allograft+chondrocytes+embedded+in+agarose+gel+into+cartilage+defects+of+rabbits.">Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits.</a> Osteoarthritis Cartilage. 6, 50-65 (1998).</li><li>Henning, T. D., Boddington, S., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+hESCs+and+hMSCs+with+iron+oxide+nanoparticles+for+non-invasive+in+vivo+tracking+with+MR+imaging.">Labeling hESCs and hMSCs with iron oxide nanoparticles for non-invasive in vivo tracking with MR imaging.</a> J Vis Exp. , (2008).</li></ol>

وصف بروتوكول يوفر وسيلة لتسمية اللجان الدائمة للتكرار مع جزيئات أكسيد الحديد وزرع هذه اللجان الدائمة في المسمى عيوب الغضروف. هذا الأسلوب يسمح غير الغازية تصوير زرع الخلايا الجذعية في عيوب الغضروف مع التصوير بالرنين المغناطيسي ، والذي يسمح للكشف المبكر عن الفشل ماز?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>D-MEM High Glucose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D5648</td>
<td>&nbsp;</td>
<tr>
<td>PBS (Ca++, Mg++ free)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14190-144</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA 0.05%</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25399-120</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Ferumoxides (Endorem)</td>
<td>
</td>
<td>Guerbet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Agarose Typ VII</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9045</td>
<td>Dilute Agarose in PBS to final concentration of 4%</td>
</tr>		</tbody>
		</table>
		</div>

implantation of ferumoxides labeled human mesenchymal stem cells in cartilage defects

مجال هندسة الأنسجة يدمج مبادئ علم الأحياء الهندسة الخلية ، والدواء نحو تجديد الخلايا والأنسجة وظيفية محددة. مصفوفة زرع الخلايا الجذعية المرتبطة به (مازي) تهدف إلى تجديد عيوب الغضروف بسبب الاصابة بالتهاب المفاصل المشتركة أو الصدمة. الكبار الخلايا الجذعية الوسيطة (MSCS) لديها القدرة على التمايز إلى خلايا النسب مكون للغضروف وأظهرت نتائج واعدة لخلية تكنولوجيات إصلاح الغضروف المفصلي. ويمكن توسيع نطاق ذاتي يمكن عزل اللجان الدائمة من مجموعة متنوعة من الأنسجة ، في ثقافات الخلية دون أن تفقد قدرتها التمايز ، وبرهنت تمايز مكون للغضروف في المختبر والمجراة في 1 و 2. من أجل توفير الاحتفاظ المحلية وصلاحية اللجان الدائمة زرع غضروف في العيوب ، لا بد من السقالة ، والذي يدعم أيضا تمايز اللاحقة والانتشار. بنية النسيج سقالة تشكيل دلائل وتصاريح المصفوفة خارج الخلية ، التي تنتجها الخلايا الجذعية ، للتوسع. وقد أظهرت التحقيقات السابقة بأن 2 ٪ agarose سقالة قد دعم تنمية مستقرة وغضروف زجاجي لا تحفز الاستجابات المناعية 3. الاحتفاظ على المدى الطويل من الخلايا الجذعية المزروعة في مازي أمر حاسم بالنسبة لتجديد الغضاريف. وسم اللجان الدائمة مع جزيئات أكسيد الحديد يسمح على المدى الطويل في تتبع المجراة مع المنظمات غير الغازية تقنيات التصوير بالرنين المغناطيسي 4. وهذا العرض شرح التقنيات لوضع العلامات اللجان الدائمة مع جزيئات أكسيد الحديد ، وتوليد خلايا agarose يبني وغرس هذه التركيبات في عيوب الغضروف. ويمكن تتبع يبني المسمى غير جراحية مع MR - التصوير.

Watch this Scientific Journal Video about Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects at JoVE.com

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

الهدف من العرض هو لاظهار طريقة استنساخه للغاية لتوليد مصفوفة الخلايا الجذعية يزرع في العيوب المرتبطة الغضروف ، والتي يمكن تصور مع التصوير بالرنين المغناطيسي. وصفت الخلايا الجذعية مع ادارة الاغذية والعقاقير التي وافق عليها Ferumoxides ، مختلطة مع agarose ، مزروع في عيوب الغضروف وتصويرها مع ماسح ضوئي 7T MR.

غرس Ferumoxides إعتبر الإنسان الخلايا الجذعية الوسيطة في عيوب الغضروف

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

implantation-ferumoxides-labeled-human-mesenchymal-stem-cells

Research

JoVE Journal

Biology

10.5K Views.  Medical Center, University of California San Francisco. الهدف من العرض هو لاظهار طريقة استنساخه للغاية لتوليد مصفوفة الخلايا الجذعية يزرع في العيوب المرتبطة الغضروف ، والتي يمكن تصور مع التصوير بالرنين المغناطيسي. وصفت الخلايا الجذعية مع ادارة الاغذية والعقاقير التي وافق عليها Ferumoxides ، مختلطة مع agarose ، مزروع في عيوب...

Video: Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

العزلة والحيوان التوسع الحرة المصل الحبل السري الإنسان المستمدة الخلايا اللحمية الوسيطة (MSCS) وغشائي خلايا مستعمرة السلف التشكيل (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

الببتيدات من مكتبة عرض بالعاثية تعدل التعبير الجيني في خلايا اللحمة المتوسطة وتحفيز تكون العظم في عيوب العظام Unicortical

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

إعادة برمجة الخلايا الجسدية البشرية المستحثة إلى الخلايا الجذعية المحفزة (iPSCs) استخدام ناقلات فيروسات الرجعية مع GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

الاستقراء وتحليل الظهارية الوسيطة الانتقالية ل

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

العزلة وثقافة الأسنان طلائي الخلايا الجذعية من الكبار ماوس القاطعة

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

كفاءة الجيل المستحثة الإنسان افرة القوة الخلايا الجذعية من الخلايا الجسدية البشرية مع سينداي الفيروسات

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

التنميط الفردية الخلايا الجذعية الجنينية البشرية من خلال الكمي RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

طريقة ترشيح بسيطة على الطاولة لعزل الحويصلات الصغيرة خارج الخلية من الخلايا الجذعية الوسيطة البشرية

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

عزل وتوصيف وتطبيق علاجي للحويصلات خارج الخلية من الخلايا الجذعية الوسيطة البشرية المستزرعة

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

عزل الخلايا الجذعية المشتقة من الأنسجة الدهنية وتوصيفها

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

تسليط الضوء على المؤلف: عزل وتحديد الخلايا الجذعية الوسيطة المشتقة من الأنسجة الدهنية لفئران Sprague Dawley  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

عزل وتحديد الخلايا الجذعية الوسيطة المشتقة من الأنسجة الدهنية لفئران Sprague Dawley

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...